This invention relates to a method of purifying proteins and nucleic acids
One of the largest contributors to false positive and false negative results in rapid detection technologies is inconsistent, variable processed samples. Sample preparation represents a critical, yet under developed capability. Present day sample preparation is usually performed manually. When automated, sample preparation involves mechanizing manual processing methods resulting in large, complex, robotic systems with significant reagent/waste streams and consumable usage.
Generic methods for the purification of DNA from cells or mixtures of cells have been available for many years and include alcohol precipitation, silica binding, standard gel electrophoresis methods, and phenol/chloroform extraction. The polymerase chain reaction (PCR), through its use of primers to amplify and detect regions of the previously-purified nucleic acid specific for different classes of organisms. The ability to develop generic purification methods for nucleic acids is due largely to the fact that all nucleic acid molecules are similar in chemical structure and these similarities can be taken advantage of when developing purification methods. However, most of these methods are time consuming and must currently be performed manually.
Proteins, on the other hand, are very different in structure from one type of protein to the next. Therefore, protein purification has largely focused on separation methods based on unique protein characteristics such as differences in size, charge, hydrophobicity, isoelectric point, antibody binding and/or enzyme-substrate specificities. When a number of these methods are performed in tandem eventually leads to a protein that has been purified away from all other proteins in the starting mixture. Typically, separation methods include chromatography electrophoresis, immunoprecipitation and magnetic separation techniques and the like. Chromatography is performed using columns which are typically quite large and require expensive equipment to obtain and analyze the samples. Therefore, methods currently used to purify nucleic acids are not used to purify protein and conversely, methods used to purify protein are not used purify nucleic acids.
U.S. Pat. No. 7,473,551 B2 (Warthoe) includes a microsensor and a hydrogel to isolate target analytes using a non-fluorescent detection system, which includes the target analytes as being nucleic acids or proteins. U.S. Pat. No. 7,371,533 discusses a method of separating polypeptides or polynucleotides using electrophoresis. U.S. Application No. 2009/0178929 (Broer et al.) discusses a device for isoelectric focusing for the separation of DNA or proteins. U.S. Application No. 2009/0139867 A1 (Marziali and Whitehead) discusses methods and an apparatus for concentrating RNA or DNA. U.S. Application No. 2008/0237044 A1 (Fiering and Keegan) discusses a method and apparatus for continuously separating or concentrating molecules that include flowing two fluids in laminar flow through an electrical field and capturing at one of three outputs a fluid stream having a different concentration of molecules. Isotachophoresis is not disclosed in any of the applications, nor is the use of TAPSO buffer.
Isotachophoresis is a technique used to separate charged particles using a discontinuous electrical field to create sharp boundaries between the sample constituents. In this method, the sample is introduced between a fast leading electrolyte and a slow moving electrolyte to create a window between which a subset of constituents from a complex matrix can be segregated from other matrix constituents. The segregation occurs after application of an electrical field to the sample and the electrolytes are allowed to separate by charge. The electrolytes used for a separation are selected experimentally so that as many contaminating constituents as possible are excluded from the final sample.
Isotachophoresis is currently used in the field of protein purification as one potential method in capillary electrophoresis for the separation of specific proteins from a mixture of proteins. However, one disadvantage of isotachophoresis in capillary use is that one can only purify either negative or positively charged ions in the capillary tubes because these moieties will migrate in different directions upon application of the electrical field. The most common use of isotachophoresis is in protein stacking gels where protein samples are added to a gel in a wide band for separation by molecular weight. However, before separation by molecular weight can occur, the protein must be concentrated into a small band to increase resolution.
U.S. Pat. No. 3,869,365 (Sunden) is drawn to a broad method of counter-flow isotachophoresis comprising two electrolytes and the flow and voltage adjusted to maintain the sample at a desired position in the column. U.S. Pat. No. 3,948,753 (Arlinger) is drawn to an apparatus for isotachophoresis comprising a column, capillary tube, a detector, and a shunt tube bifurcating the column. U.S. Pat. No. 6,685,813 B2 (Williams et al.) is drawn to a method of separating components using isotachophoresis. U.S. Appl. No. 2004/0060821 A1 (Williams et al.) is drawn to a method of separating components using isotachophoresis. U.S. Pat. No. 7,494,577 B2 and U.S. Application No. 2004/0060821 (Williams et al.) discusses a method of separating components by loading a microchannel with a sample, placed between a trailing-edge electrolyte and a leading-edge electrolyte using isotachophoresis. There is no disclosure of the separation of either nucleic acids or proteins or to a TAPSO buffer. Application No. WO 2008/025806 (Gerhard Weber) is directed to a method of separating particles using electrophoresis and discloses isotachophoresis. Baumann, G. and Chrambach, A. disclosed the use of isotachophoresis for the isolation of hormones. (Proc. Natl. Acad. Sci. USA, 73(3): 732-736, (1976)). Böttcher, A. et al. disclosed preparative isotachophoresis for separation of human plasma lipoproteins, apolipoproteins and HDL subfractions, (J. Lipid Res. 41:905-915, (2000)). None disclose the simultaneous separation and purification of both nucleic acids and proteins. U.S. Appl. No. 2004/0031683 A1 (Eipel et al.) is drawn to a method of fractionating proteins using several procedures, including isotachophoresis. The two-step process includes capillary electrophoresis, but does not disclose the separation of nucleic acids. U.S. Pat. No. 7,399,394 B2 (Gerhard Weber) is directed to a free flow electrophoresis (FFE) (also known as carrierless electrophoresis or isotachophoressis) method and related devices. U.S. Pat. No. 5,817,225 (Hinton) is drawn to an electrophoresis unit comprising an anode compartment, a cathode compartment, a separation chamber and an electrolyte in said chamber with an electrophoretic mobility between the mobilities of the nucleic acids and organic salts which are to be separated. The separation of proteins was not disclosed. U.S. Pat. Nos. 7,316,771 B2 and 7,247,224 (withdrawn) (both by Gerhard Weber) is directed to a medium for electrophoresis comprising at least two acids and at least two bases as buffers. Neither disclose the separation of nucleic acids or proteins. U.S. Pat. No. 6,780,584 B1 (Edman et al.) is broadly drawn to a device with a first buffer reservoir containing a first buffer with a differing conductances, a conductive semipermeable matrix, and a first and second electrode and a specific binding entity and discloses a device which separates nucleic acid and RNA in hybridization reactions, but not the separation of proteins. U.S. Pat. No. 5,464,515 (Bellon) is drawn to a procedure for loading one of several biological samples on an electrophoresis slab support, but does not disclose the separation of nucleic acids. U.S. Pat. No. 7,214,299 B2 (Armstrong) is broadly directed to the separation of microbes and cells using electrophoresis methods. U.S. Pat. No. 5,447,612 (Bier et al.) is directed to a buffering composition for electrophoresis methods, including TAPSO and EACA (epsilon-aminocaproic acid) as complementary buffer pairs and methods. comprising the buffer composition in an isoelectric focusing method, used with a recycling focusing instrument of the '169 Bier patent. 2008/0197019 A1 (Santiago and Khurana) discloses a method of using an electric field to isolate proteins and nucleic acids. Isotachophoresis is not disclosed, nor is the use of TAPSO buffer. 2010/0029915 discloses automated methods to isolate proteins or nucleic acids comprising the use of BES buffer and does not mention ITP. Xu, Z Q et al., (J. Chromatography, A, 1216(4):659-670, (January 2009)) discusses electrokinetic supercharging as a transient ITP including the separation of proteins and nucleic acids.
Appl. No. EP 05076569.2 (Stichting voor de Technische Wetenschnappen) is drawn to a device for separating particles and the use of isotachophoresis for the non-simultaneous separation of nucleic acids or proteins and the use of binding molecules. Blessum, C. et al. disclose capillary electrophoresis in the separation of proteins, nucleic acids and lipoproteins, and its use in isotachophoresis. (Ann. Biologie Clinique, 57(6):643-647 (1999) [French]). Blessum et al. do not disclose the simultaneous isolation of nucleic acids and proteins using isotachophoresis. Dolnik, V. et al. disclose capillary electrophoresis techniques and microchip technology which includes isotachophoresis, and current methods of separation of nucleic acids or proteins, but does not disclose simultaneous separation of nucleic acids and proteins (Electrophoresis, 21(1):41-54, (1999) [Abstract only]). PCT Application No. WO 2008/082876 A1 (Balgley) is directed to a method of separating DNA or protein from a heterogeneous biomolecular sample using isotachophoresis coupled with liquid column chromatography. PCT Application No. WO 2008/053047 A2 (Weber, Gerhard) is directed to an electrophoresis method comprising a spacer zone and also separating at least one analyte, including DNA, protein or protein complexes using isotachophoresis. PCT Application No. WO 2007/008064 A1 (Kohlheyer et al.) is directed to a device and use of the device for separating particles in a fluid sample utilizing free flow isotachophoresis; and a discussion of the separation of DNA and proteins, although there is no mention of simultaneous separation. U.S. Pat. No. 4,897,169 (Bier and Twitty) discloses isotachophoresis and an apparatus using TRIS and cacodylic acid as electrolyte components. Hirukawa, T. et al. discusses the use of ITP for the separation of DNA fragments and SDS-proteins, but not simultaneously. (Bunseki Kagaku, (Japanese language, Abstract and FIGS. 13 and 16) 52(12):1069-1079 (2003)). Khurana, T. K. et al. describe ITP separation of DNA and proteins (Lab Chip, 9:1377-1384 (March 2009)). U.S. Patent Publication No. 2009/0032401 A1 (Ronaghi, Khurana, and Santiago) discloses a method of using an electric field to isolate proteins and nucleic acids, concentrating and separating at least one directly undetectable analyte of interest and said at least two directly detectable spacer molecules into zones using isotachophoresis; the analytes can be DNA or RNA. Lin, C-C et al., discusses an integrated ITP-gel electrophoresis device and discusses the separation of DNA and protein molecules. (Electrophoresis, 29(6):1228-1236, (March 2008)). Shackman, J. G and Ross, D. discusses a capillary isotachophoresis method and discusses the separation of DNA or protein molecules (Anal. Chem., 79(17):6641-6649, (August 2007)).
Newer, rapid, simpler methods of sample processing are required to support the next generation detection systems. The newer detection methods will have a heavier reliance on sample preparation for the generation of meaningful results. In addition, sample preparation methods will need to support numerous different detection systems and be capable of being integrated as part of a complete sample processing and detection system.
Therefore, there is currently a great need in sample processing methods that are fast, inexpensive, and are easy to perform, even by untrained, non-technical staff in a variety of disciplines including biodefense, food and water, agricultural, environmental, clinical testing, and the like.